Specimen processing systems and related methods

ABSTRACT

A specimen processing system includes a plate for supporting a specimen system, wherein the specimen system includes a container and a specimen contained therein. The specimen processing system further includes a camera disposed above the plate and configured to generate images of the specimen system, a light source disposed beneath the plate for radiating light towards the plate, a light stop for blocking a portion of the light from reaching the specimen system to produce darkfield illumination of the specimen at the camera, and one or more processors electronically coupled to the camera and configured to track a position of the specimen within the specimen container during a specimen processing protocol based on the images.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of prior U.S. ProvisionalApplication No. 62/894,202, filed on Aug. 30, 2019, which isincorporated herein by reference in its entirety.

TECHNICAL FIELD

This disclosure relates to specimen processing systems, such asautomated vitrification systems, and methods of tracking a position of aspecimen within a specimen container undergoing a specimen processingprotocol at such specimen processing systems.

BACKGROUND

Cryopreservation containers are used in the field of assistedreproductive technology (ART) to store and preserve living reproductivespecimens (e.g., oocytes, embryos, and blastocysts). Cryopreservationrefers to a process in which specimens are preserved over extendedperiods of time by cooling to sub-zero temperatures. For example, acryopreservation container can house and support specimens undergoingvitrification, which is the rapid transition of a substance from aliquid phase to a solid phase (e.g., glass) without the formation of icecrystals within cells of the specimen. Typical protocols for vitrifyinga reproductive specimen include exposing the specimen to multipleprocessing solutions according to a detailed vitrification protocol,subsequently transferring the specimen to a cryopreservation container,and then exposing the cryopreservation container, containing thespecimen therein, to a low temperature cooling medium (e.g., liquidnitrogen) to cause the cells of the specimen to rapidly cool to a glassstate before ice crystals can form within the cells. Thecryopreservation container can be stored in the cooling medium until thespecimen is ready to be used in reproductive procedures.

SUMMARY

In general, this disclosure relates to specimen processing systems thatcan be used to prepare a biological specimen for cryopreservation withina specimen container according to a specimen processing protocol (e.g.,a vitrification protocol) in an automated manner.

In one aspect, a specimen processing system includes a plate forsupporting a specimen system, wherein the specimen system includes acontainer and a specimen contained therein. The specimen processingsystem further includes a camera disposed above the plate and configuredto generate images of the specimen system, a light source disposedbeneath the plate for radiating light towards the plate, a light stopfor blocking a portion of the light from reaching the specimen system toproduce darkfield illumination of the specimen at the camera, and one ormore processors electronically coupled to the camera and configured totrack a position of the specimen within the specimen container during aspecimen processing protocol based on the images.

Embodiments may include one or more of the following features.

In some embodiments, the specimen processing system further includes anadjustable lens for focusing the light onto the specimen system.

In some embodiments, the specimen processing system further includes aprocessing station that locates the camera.

In some embodiments, the processing station defines a receptacleadjacent the plate for positioning the specimen container.

In some embodiments, the processing station includes a mount forselectively positioning the camera at the processing station.

In some embodiments, the specimen processing system further includes arotatable platform to which the processing station is secured forapplying a centripetal force to the specimen to cause the specimen tomove within the specimen container.

In some embodiments, the one or more processors are further configuredto convert the images from color to greyscale.

In some embodiments, the one or more processors are further configuredto remove noise from the images.

In some embodiments, the one or more processors are further configuredto detect an object corresponding to the specimen in the images.

In some embodiments, the one or more processors are further configuredto determine parameters including one or more of a position, a speed,and a direction of the specimen as the specimen moves within thespecimen container.

In some embodiments, the one or more processors are configured to outputone or more of the parameters.

In some embodiments, the specimen processing system further includes amotor that can adjust movement of the rotatable platform based on one ormore of the parameters.

In some embodiments, the light stop is arranged to block the portion ofthe light from reaching a central axis of the specimen container suchthat edges of the specimen remain visible to produce darkfieldillumination at the camera.

In some embodiments, the light source includes multiple light-emittingdiodes.

In some embodiments, the camera is configured to scan an identificationlabel of the specimen container.

In some embodiments, the one or more processors are configured to trackrespective positions of multiple specimens within the specimen containerbased on the images during the specimen processing protocol.

In some embodiments, the specimen processing system further includes avibration assembly configured to direct movement of the specimen withinthe specimen container during the specimen processing protocol.

In some embodiments, the specimen processing system further includes acutting station configured to cut and release a distal portion of thespecimen container with the specimen contained therein followingcompletion of the specimen processing protocol.

In some embodiments, the specimen is a reproductive specimen.

In some embodiments, the specimen processing protocol includes avitrification protocol.

In another aspect, a method of processing a specimen within a specimencontainer includes generating images of the specimen within the specimencontainer at a camera disposed above a plate supporting the specimencontainer, directing light towards the plate from a light sourcedisposed beneath the plate, blocking a portion of the light fromreaching the specimen with a light stop to produce darkfieldillumination of the specimen at the camera, and tracking a position ofthe specimen within the specimen container based on the images at one ormore processors in electronic communication with the camera during aspecimen processing protocol.

Embodiments, may include one or more of the following features.

In some embodiments, the method further includes focusing the light ontothe specimen at an adjustable lens.

In some embodiments, the method further includes locating the camera ata processing station.

In some embodiments, the method further includes positioning thespecimen container within a receptacle of the processing station that isadjacent the plate.

In some embodiments, the method further includes selectively positioninga mount supporting the camera at the processing station.

In some embodiments, the method further includes applying a centripetalforce to the specimen to cause the specimen to move within the specimencontainer by rotating a platform to which the processing station issecured.

In some embodiments, the method further includes converting the imagesfrom color to greyscale at the one or more processors.

In some embodiments, the method further includes removing noise from theimages at the one or more processors.

In some embodiments, the method further includes detecting an objectcorresponding to the specimen in the images at the one or moreprocessors.

In some embodiments, the method further includes determining, at the oneor more processors, parameters including one or more of a position, aspeed, and a direction of the specimen as the specimen moves within thespecimen container.

In some embodiments, the method further includes outputting one or moreof the parameters from the one or more processors.

In some embodiments, the method further includes adjusting movement ofthe platform based on one or more of the parameters via a motor.

In some embodiments, the method further includes blocking the portion ofthe light from reaching a central axis of the specimen container suchthat edges of the specimen remain visible to produce darkfieldillumination at the camera.

In some embodiments, the light source includes multiple light-emittingdiodes.

In some embodiments, the method further includes scanning anidentification label of the specimen container at the camera.

In some embodiments, the method further includes tracking respectivepositions of multiple specimens within the specimen container based onthe images at the one or more processors during the specimen processingprotocol.

In some embodiments, the method further includes directing movement ofthe specimen within the specimen container at vibration assembly duringthe specimen processing protocol.

In some embodiments, the method further includes cutting and releasing adistal portion of the specimen container, with the specimen containedtherein, following completion of the specimen processing protocol at acutting station.

In some embodiments, the specimen is a reproductive specimen.

In some embodiments, the specimen processing protocol includes avitrification protocol.

Embodiments may provide one or more of the following advantages.

In some embodiments, the specimen processing system includes one or moreprocessing stations that are configurable owing to multiple mounting andsupport components for particularly positioning the specimen containeras desired. The specimen processing system also includes amicrocontroller that can advantageously adjust a rotational speed of aplatform on which the specimen container rotates and a duration of oneor more phases of a specimen processing protocol based on feedback froma vision system.

For example, in some embodiments, a vision system located at eachprocessing station is configured to provide darkfield illumination ofthe specimen for optimal visualization and tracking of the specimenduring the specimen processing protocol. The configuration andfunctionality of the various components of the vision system forachieving dark field illumination advantageously allow for fine controland constraint of intensity, exposure time, and wavelength of lightradiating from a light source to the specimen, which can be important tothe survival of delicate biological specimens.

Furthermore, in some embodiments, a camera of the vision system cantrack a linear movement of the specimen throughout the specimenprocessing protocol in real time by continuously generating images ofthe specimen and feeding the images in a real-time video feed to acomputing device running a software algorithm that processes the imagesto track a position of the specimen. Based on feedback from the softwarealgorithm, the microcontroller advantageously can control the rotationalspeed, spin direction, and acceleration of the platform to ensure thatthe specimen is exposed to a substantially constant centripetal force asprogrammed by the user. Such protocol adjustments can optimize timeperiods of specimen exposure to the processing media.

DESCRIPTION OF DRAWINGS

FIG. 1 is a front perspective view of a specimen processing system thatcan be used to prepare a specimen disposed within a specimen container.

FIG. 2 is a side perspective view of the specimen processing system ofFIG. 1 .

FIG. 3 is a rear perspective view of the specimen processing system ofFIG. 1 .

FIG. 4 is a top perspective view of the specimen processing system ofFIG. 1 with certain components of a processing station omitted.

FIG. 5 is a side view of a specimen container that can be processed atthe specimen processing system of FIG. 1 .

FIG. 6 is a cross-sectional view of a proximal end region of thespecimen container of FIG. 5 , including an identification (ID) labelprovided as an RFID tag.

FIG. 7 is a cross-sectional view of a proximal end region of thespecimen container of FIG. 5 , including an ID label provided as abarcode tag.

FIG. 8 is a cross-sectional view of a proximal end region of thespecimen container of FIG. 5 , including an ID label provided as a QRcode tag.

FIG. 9 is a front perspective view of the specimen processing system ofFIG. 1 with certain portions of a housing omitted to expose certaininternal components.

FIG. 10 is a bottom perspective view of the specimen processing systemof FIG. 1 with certain portions of a housing omitted to expose certaininternal components.

FIG. 11 is a front perspective view of a platform and certain otherassociated components of the specimen processing system of FIG. 1 .

FIG. 12 is a top perspective view of the platform of FIG. 11 .

FIG. 13 is an exploded perspective view of a vision system of thespecimen processing system of FIG. 1 .

FIGS. 14-18 illustrate a series of movements of a specimen within thespecimen container of FIG. 1 for processing the specimen according to aprotocol carried out at the specimen processing system of FIG. 1 .

FIG. 19 illustrates a flowchart of a software algorithm that processesimages of a specimen during a protocol carried out at the specimenprocessing system of FIG. 1 .

FIG. 20 is a perspective view of vibration assembly of the specimenprocessing system 100.

FIG. 21 is a side view of a cut-and-seal station of a specimenprocessing system.

FIG. 22 is a side view of a cutting station of a specimen processingsystem.

DETAILED DESCRIPTION

FIGS. 1-4 illustrate various views of a specimen processing system 100that can be used to prepare a biological specimen for cryopreservationwithin a specimen container 1000 according to a specimen processingprotocol (e.g., a vitrification protocol) in an automated manner.Referring to FIG. 5 , the specimen 1001 is disposed within the specimencontainer 1000, and the specimen container 1000 is designed forcryopreparation and cryopreservation of the specimen 1001 in a viableand vitrified state within a low temperature substance (e.g., liquidnitrogen, cryogenic plasma, or liquid helium) until the specimen 1001 isdesired for use (e.g., over a period of up to about 30 years). Thespecimen 1001 may be a single cell, a collection of free (e.g.,unattached) cells, or a collection of attached cells (e.g., amulticellular tissue). The specimen 1001 may be a reproductive specimen(e.g., a sperm cell, an oocyte, a zygote, a blastocyst, a gastrula, oran embryo) or a non-reproductive specimen (e.g., one or more T-cells orblood cells). The specimen 1001 may be a mammalian tissue sample or anon-mammalian tissue sample. In some examples, the specimen 1001 may bean agricultural specimen, such as canola. In other examples, thespecimen 1001 may be a non-biological specimen, such as variouschemicals or other non-biological specimens.

The specimen processing system 100 and the specimen container 1000 aretogether designed to exploit mass properties (e.g., density and fluidmechanics) of the specimen 1001 with respect to mass properties ofvarious processing media. Accordingly, the specimen container 1000 isprovided as an elongate tube 1002 that is internally preloaded withmultiple fluids to which the specimen 1001 will be exposed during acryopreservation process. In particular, the specimen 1001 can be movedin an axial direction 1003 within the specimen container 1000 bycentrifugal forces acting on the specimen 1001 within the processingsystem 100, as will be discussed in more detail below.

The elongate tube 1002 is hermetically sealed at proximal and distalclosures 1004, 1006. In some embodiments, the elongate tube 1002 ispreloaded with an equilibration solution 1008 (e.g., a cryoprotectant ofrelatively low density) and a vitrification solution 1010 (e.g., acryoprotectant of relatively high density) that are separated by aseparation fluid 1012 (e.g., an air bubble or an immiscible media). Suchseparation of the equilibration solution 1008 and the vitrificationsolution 1010 enables appropriate processing of the specimen 1001 (e.g.,sequential exposure of the specimen 1001 to particular solutions fordesired periods of time) during a vitrification protocol. In someembodiments, the elongate tube 1002 is further preloaded with a proximalair pocket 1014 that separates the equilibration solution 1008 from theproximal closure 1004 and a distal air pocket 1016 (e.g., occupying aportion of an interior volume of a tapered portion 1018 of the elongatetube 1002) that separates the vitrification solution 1010 from thedistal closure 1006.

The elongate tube 1002 is a thin capillary tube of very small diameter(e.g., having an internal diameter on the order of 10⁻⁴ m). The elongatetube 1002 has a substantially constant diameter along a main portion1020 (e.g., a cylindrical portion) and has a variable diameter thatgradually decreases along the tapered portion 1018. A lumen of theelongate tube 1002, at a smallest inner diameter, is large enough toaccommodate a specimen 1001, which typically has a diameter or a widthin a range of about 50 μm to about 150 μm. The specimen container 1000typically has a total length of about 15 mm to about 260 mm (e.g., about150 mm). The elongate tube 1002 is typically made of one or morematerials that are transparent or translucent to allow viewing of thespecimen 1001 contained within the elongate tube 1002 and that canwithstand the low temperature substance. Example materials from whichthe elongate tube 1002 may be made include polymers such as polystyrene,polypropylene, polyvinyl acetate, and polycarbonate, and fluoropolymers.

Referring to FIGS. 6-8 , the specimen container 1000 further includes,respectively, an identification (ID) label 1022, 1024, or 1026 attachedto the elongate tube 1002 near the proximal closure 1004. The ID labelmay be attached to the elongate tube 1002 with a self-adhesive stickeror embedded within the wall of the elongate tube 1002. The ID labelincludes machine readable information and may additionally include humanreadable information that is written on an outer surface of the IDlabel. Either or both of the machine readable information and the humanreadable information may include various patient data, such as a name, abirthdate, a unique reference code (e.g., an alphanumeric sequence), andother patient data. The ID label of the specimen container 1000 can bedetected and read by a scanning component of the specimen processingsystem 100, as will be discussed in more detail below. As shownrespectively in FIGS. 6-8 , the ID label may be embodied as aradio-frequency identification (RFID) tag 1022 (e.g., including aninternal antenna), a barcode 1024 tag (e.g., including a one-dimensionalcode format), or a quick response (QR) code 1026 tag (e.g., including atwo-dimensional code format).

Referring to FIGS. 1, 2, and 4 , the specimen processing system 100 isprovided as a console that includes multiple processing stations 102 atwhich respective specimen containers 1000 can be secured to carry outthe specimen processing protocol, a platform 104 along which theprocessing stations 102 are disposed, a housing 106 that enclosesinternal components located beneath the platform 104, handles 188 forlifting or otherwise moving the specimen processing system 100, and alid 108. The lid 108 is openable from the housing 106 to permit accessto the processing stations 102 and closeable upon the housing 106 toprevent access to or otherwise protect the processing stations 102. Thespecimen processing system 100 further includes a display screen 110 forpresenting various user interfaces, multiple selectors 112 (e.g.,buttons) for setting various operational parameters of the specimenprocessing system 100 and process parameters of the specimen protocol, apower switch 192, and a cable port 114 that are positioned along a frontwall of the housing 106, and a power connector 116 that is positionedalong a rear wall of the housing 106.

The housing 106 is designed to rest atop a table surface, a floorsurface, or another flat surface. The housing 106 defines air vents 118positioned along lateral walls and air vents 120 positioned along therear wall. The air vents 118 allow air to circulate into and out of thehousing 106 to prevent internal components disposed within the housing106 from exceeding a threshold temperature of about 80° C. The housing106 also defines a power connector 122 along the rear wall. The housing106 is connected to the lid 108 via hinges 124.

In some embodiments, the housing 106 and the lid 108 of the specimenprocessing system 100 together have a total length of about 0.2 m toabout 1.0 m, a total width of about 0.2 m to about 1.0 m, and a totalheight of about 0.2 m to about 1.0 m. In some embodiments, the specimenprocessing system 100 has a weight in a range of about 5 kg to about 50kg and is typically stored on a laboratory floor, a storage facilityfloor, a table, or a countertop, that has an ambient environmentaltemperature of about 18° C. to about 28° C. In some embodiments, areceptacle 162 of a processing station 102 has a length of about 5 cm toabout 15 cm and a width of about 1 cm to about 5 cm. The housing 106 andthe lid 108 are typically made of materials that provide a significantdegree of thermal insulation, such as polymers.

Additionally, the specimen processing system 100 includes a timer 126for tracking durations of various phases of the specimen processingprotocol, a reader component 128 that is programmed to read ID labels ofspecimen containers 1000, and a microcontroller 130 that is programmedto control various features and functionalities of the specimenprocessing system 100. The timer 126, the reader component 129, and themicrocontroller 130 (all illustrated schematically in FIG. 1 ) may belocated at positions that are suitable for their respective functions.For example, any of the timer 126, the reader component 129, and themicrocontroller 130 may be mounted on any sidewall of the housing 106(e.g., a base portion, a lateral portion, a top portion, or a bottomportion) or a support member attached thereto]. For example, in someembodiments, the microcontroller 130 may be located adjacent the displayscreen 110.

The display screen 110 allows a user to input several parameters thatgovern operation of the specimen processing system 100 to process (e.g.,vitrify) one or more specimens 1001. In some examples, such inputparameters are related to a specimen 1001, such as a developmental stageof the specimen 1001 (e.g., resulting in a selection of an oocyteprotocol or a blastocyst protocol). The display screen 110 may be anintegrated touchscreen or a touchless screen associated with tactilecontrol elements, such as buttons, knobs, dials, or the like.

The microcontroller 130 includes one or more processors that are incommunication with and/or are programmed to control various actuatorsand sensors of the specimen processing system 100 related to variousautomated features, such as receiving and instantiating user selectionsinput at the display screen 110, reading an ID label of a specimencontainer 1001, executing the timer 126, spinning the platform 104 at aspecified spin speed for a specified duration, detecting an open orclosed state of the lid 108, and providing audible and/or visualfeedback regarding a progression of the specimen processing protocol. Insome embodiments, the platform 104 can only be activated to spin oncethe lid 108 is closed and interlocked with the housing 106. Furthermore,once the platform 104 is spinning as part of a specimen processingprotocol, the lid 108 may not be openable until spinning of the platform104 has ceased.

Referring particularly to FIG. 4 , the platform 104 defines multiple(e.g., six) slots 132 at which a processing station 102 can be secured(e.g., bolted) to the platform 104 in a fixed position. The slots 132are formed as elongate openings along which a specimen container 1000can be aligned and therefore define multiple, optional locations atwhich a specimen container 1000 can be positioned on the platform 104.Each slot 132 is flanked by a set of four holes 134 and two sets of twoholes 136 that are distributed in arrangements that are parallel to theslot 132. A processing station 102 can therefore be attached to theplatform 104 at the holes 134, 136 for examination of a specimen 1001inside of a specimen container 1000 positioned along the slot 132.According to an arrangement of the multiple slots 132, sizes of thevarious components of a processing station 102, and functionalrequirements of the specimen processing system 100 (e.g., maintaining asubstantially balanced mass across the platform 104 during a protocol),only two or three processing stations 102 may be installed to theplatform 104 at any given time in some examples, and the two or threeprocessing stations 102 should be spaced circumferentially,substantially equally apart from one another about the platform 104. Inother examples, a different number and spacing of processing stations102 may be implemented, as long as a method of balancing mass across theplatform 104 is employed, such as by strategically placingcounterweights along the platform 104.

Referring to FIGS. 9 and 10 , in which certain portions of the housing106 and the lid 108 have been omitted to expose certain interiorfeatures, the specimen processing system 100 further includes a printedcircuit board (PCB) 138 and a motor assembly 140 that are assembled withthe platform 104 and a PCB 154 that is positioned along a front wall(omitted from FIGS. 9 and 10 for clarity) of the housing 106. In someembodiments, the timer 126 and the microcontroller 130 (illustratedschematically in FIG. 1 ) are implemented at the PCB 154. An assembly ofthe platform 104 and the motor assembly 140 ensures fast and smoothacceleration between rotational speed changes of the platform 104.

Referring particularly to FIG. 10 , the PCB 138 is attached (e.g.,bolted) to a bottom surface of the platform 104 and includes multiple(e.g., six) extension plates 142 that are sized, positioned, andoriented to align with the multiple slots 132 of the platform 104. Amatrix (e.g., two arrays) of multiple light emitting diodes (LEDs) 144are mounted to an upper surface of each extension plate 142 and areexposed through the slots 132 of the platform 104 (refer to FIG. 4 ).The motor assembly 140 includes a rotatable motor block 190, a supportplate 146 attached to an upper surface of the motor block 190, supportcolumns 148 that extend from the support plate 146 to the platform 144,and a cylindrical coupling unit 150 that extends from the motor block190 (e.g., through the support plate 146) to the platform 104. The motorblock 190 may be a servo motor or a stepper motor with an attachedencoder to provide continuous monitoring of motor speed and positionsuch that specific commands can be executed to move the platform 104 tospecific positions as desired for carrying out various actions (e.g.,mounting or dismounting a specimen container 1000 from the specimenprocessing system 100). The cylindrical coupling unit 150 is attached toboth the platform 104 and the motor block 190, such that rotation of themotor block 190 causes rotation of the coupling unit 150 and rotation ofthe platform 104 about a central axis 152 of the platform 104.Additionally, the specimen processing system 100 also includes a motorpower supply and heat sink 196 and a power converter 198 that convertssource electricity (e.g., 110 Volts/220 Volts) for some, or all, of thecomponents requiring electricity in the specimen processing system 100.The cylindrical coupling unit 150 is equipped with multiple cylindricalelectrical contacts 156 (e.g., slip rings) that transmit data andcontrol signals among the processing stations 102, the motor block 190,and the microcontroller 130.

Referring to FIGS. 11 and 12 , each processing station 102 includes alower bracket 158 and an upper bracket 160 that together define areceptacle 162 for holding a specimen container 1000 along a slot 132 ofthe platform 104. In some embodiments, the processing station 102further includes one or more spring-loaded retaining strips or clampsthat help to secure the specimen container 1000 within the receptacle162. The lower bracket 158 defines holes 164 and holes 166 that arepositioned to be aligned with one or more of the holes 134 and one ormore of the holes 136 for attaching the processing station 102 to theplatform 104 along a particular slot 132. The lower bracket 158 alsodefines multiple (e.g., four) flanges 168 that secure the upper bracket160 to the lower bracket 158. Each processing station 102 also includesa post 170 that passes through alignment holes defined respectively bythe upper and lower brackets 160, 158 to ensure a correct positioning ofthe upper bracket 160 along the lower bracket 158. The lower bracket 158further defines oppositely disposed, raised slots 176 and lateralthrough channels 178. The upper and lower brackets 160, 158 of theprocessing station 102 and the platform 104 are typically made of one ormore metals, such as aluminum, magnesium, stainless steel, and othermetals.

Each processing station 102 further includes a camera 180 by whichmovement of a specimen 1001 within a specimen container 1000 can beobserved, a mounting bracket 182 that supports the camera 180, and acover plate 194 for containing the camera 180 within the mountingbracket 182. The mounting bracket 182 defines two oppositely disposedelongate projections 184 that are sized and positioned to slide withinthe raised slots 176 to position the camera 180 at a desired locationalong the lower bracket 158. The mounting bracket 182 further definestwo sets of oppositely disposed holes 186 along the projections 184 thatcan be selectively aligned with the through channels 178 to secure themounting bracket 182 to the lower bracket 158 at the desired location.

When the specimen 1001 is to be processed within the specimen container1000 at the specimen processing system 100, an operator inputs detailedinformation about the specimen 1001 at the display screen 110, or suchinformation may be automatically imported into the specimen processingsystem 100 from another device through a data connection. In someembodiments for which the specimen container 1000 is not pre-equippedwith an ID label (e.g., an ID label 1022, 1024, or 1026), the specimenprocessing system 100 may be configured to print human readableinformation or a barcode onto an ID label using the automaticallyimported information and further attach the ID label to the specimencontainer 1000, or the printed ID label may then be manually attached tothe specimen container 1000 by the operator.

In any case, once the detailed information about the specimen 1001 isinputted manually or imported automatically, the operator then loads thespecimen container 1000, equipped with the ID label, into a receptacle162 at a processing station 102. The reader component 128 can detect apresence of the specimen container 1000 within the receptacle 162 byreading the ID label and can communicate such detection to themicrocontroller 130. In some embodiments, the reader component 128 maybe a feature of the camera 180. For example, if the ID label is providedas a barcode label 1024 or as a QR code label 1026, then the camera 180may be configured and programmed to read such label.

If the manually inputted or automatically imported information does notmatch the information that the reader component 128 reads from the IDlabel, then the specimen processing system 100 generates and displays anerror on the display screen 110 and prevents activation of a specimenprocessing protocol. If the manually inputted or automatically importedinformation does match the information that the reader component 128reads from the ID label, then the specimen processing system 100 cancause the timer 126 to be activated for processing the specimen 1001according to a specified protocol. According to one or more signalsreceived from the microcontroller 130, the platform 104 can spin aboutthe central axis 152 to exert enough centripetal force on the specimen1001 to cause the specimen 1001 to move along the axial direction 1003within the specimen container 1000 toward the distal closure 1006 (referto FIG. 5 ) according to the protocol. While the platform 104 isspinning, the specimen 1001 and the various processing media (e.g., theequilibration and vitrification solutions 1008, 1010, and any othermedia) within the specimen container 1000 can be visualized (e.g.,imaged) by the camera 180. In some embodiments, one or more parametersof the protocol may be determined by or associated with the type of IDlabel (e.g., RFID, bar code, or QR code) present on the specimencontainer 1000.

The microcontroller 130 can adjust either or both of a rotational speedof the platform 104 and a duration of one or more phases of the protocolbased on feedback from a vision system (e.g., including the camera 180)regarding an axial position of the specimen 1001, as will be discussedin more detail. Such protocol adjustments can optimize time periods ofspecimen exposure to the processing media within the specimen container1000. Upon completion of the processing protocol, the specimen container1000 may be removed from the receptacle 162 and placed within a lowtemperature substance for vitrification and cryopreservation of thespecimen 1001 contained within the specimen container 1000.

As discussed above, a camera 180 can be used to track a position of aspecimen 1001 within the specimen container 1000 during a specimenprocessing protocol. As shown in FIG. 13 , each camera 180 is acomponent of a vision system 200 located at each processing station 102of the specimen processing system 100. In addition to a camera 180, eachvision system 200 further includes an optically clear plate 202 on whichthe specimen container 1000 can be supported, upper and lower lenses204, 206 (e.g., plano-convex lenses), two adjustment screws 208 thatextend between the upper and lower lenses 204, 206, two compressionsprings 210 that respectively surround the adjustment screws 208, alight source 212 that includes an extension plate 142 of the PCB 138 anda matrix (e.g., one or more arrays) of LEDs 144 distributed along theextension plate 142, and an opaque light stop 214 that blocks centrallydirected light rays (e.g., light rays directed substantially towards acentral axis 1028 of the specimen container 1000) from impinging on thespecimen container 1000. In some embodiments, a plate 202 of a visionsystem 200 may be disposed within the a slot 132 of the platform 104.The camera 180 is typically located at a distance of about 1 cm to about5 cm above the platform 104.

The upper and lower lenses 204, 206 are focusing lenses that cancollimate light radiating from the LEDs 144 into a light beam and focusthe light beam onto an expected path of the specimen 1001 (e.g.,generally along the central axis 1028 of the specimen container 1000).Accordingly, the adjustment screws 108 and the surrounding compressionsprings 210 allow a height adjustment of the upper and lower lenses 204,206 such that the focal point of the light beam coincides with a heightof the support plate 202 on which the specimen container 1000 is held.The support plate 202 is typically positioned at a distance of about 0.1cm to about 1.5 cm above the light source 212.

The light stop 214 blocks centrally directed light rays from the LEDs144 such that when the upper and lower lenses 204, 206 are focusedcorrectly, peripheral edges (e.g., located off-axis) of the specimen1001 are illuminated. Therefore, the peripheral edges of the specimen1001 appear brighter than an interior region of the specimen 1001 tomake the specimen 1001 more visible to the camera 180 in a mannersimilar to that of dark field illumination. Furthermore, the visionsystem 200 may include a filtering functionality that blocks light withwavelengths of less than about 500 nm from reaching the specimen 1001,as exposure to such wavelengths over the extended period of specimentracking may be detrimental to the health and subsequent biologicaldevelopment of the specimen 1001. Accordingly, the configuration andfunctionality of the various components of the vision system 200 forachieving dark field illumination advantageously allow for fine controland constraint of intensity, exposure time, and wavelength of lightradiating from the light source 212 to the specimen 1001, which can beimportant to the survival of the delicate biological specimen 1001.

The camera 180 can track a linear movement of the specimen 1001throughout a specimen processing protocol in real time by continuouslygenerating images of the specimen 1001 and feeding the images at regularintervals or in the form of a real-time video feed wirelessly to aremote computing device running a software algorithm 300 (refer to FIG.19 ) that processes the images to track a position of the specimen 1001or through a wired connection via the electrical contacts 156 to one ormore processors of the microcontroller 130 running the softwarealgorithm 300. Referring to FIG. 14 , the specimen container 1000 isallowed to sit in place (e.g., stationary) in the receptacle 162 for afirst predetermined exposure period during the specimen processingprotocol so that the specimen 1001 can equilibrate in the equilibrationsolution 1020. The first exposure period may range from about 5 minutesto about 15 minutes, depending on various parameters of typical ARTprotocols.

During the first exposure period, the equilibration solution 1020 drawswater molecules out from the specimen 1001 and infuses cryoprotectantsinto the specimen 1001 according to osmotic potential. The reduction ofwater content and addition of cryoprotectants aids in minimizing damageto cellular components of the specimen 1001 during freeze and warmingcycles. Although the specimen 1001 is denser than the equilibrationsolution 1020 and will therefore very gradually descend through theequilibration solution 1020 due to gravitational forces over time, thespecimen 1001 will typically still be suspended within the equilibrationsolution 1020 and will not have yet reached the separation fluid 1024 bythe end of the first exposure period, as shown in FIG. 14 .

Referring to FIGS. 15-18 , once the specimen 1001 has been exposed tothe equilibration solution 1020 for the predetermined exposure period,the platform 104 is activated to spin the specimen container 1000 at aselect low speed to advance the equilibration solution 1020 and thespecimen 1001 axially through the separation fluid 1024 to thevitrification solution 1022. The specimen container 1000 is typicallyspun for about 0.5 minutes to about 5 minutes at an angular speed ofabout 50 rpm to about 1200 rpm, which exerts enough centripetal force onthe specimen 1001 to cause the specimen 1001 to descend into thevitrification solution 1022 in a timely manner, but not enough to causemechanical damage to the specimen 1001. Such speed (e.g., correspondingto about 5 g to about 200 g) is significantly slower than speeds of evenvery low-speed conventional laboratory centrifuges, which are typicallycapable of revolving specimens about a centrifuge axis at speeds in arange of about 4000 rpm to about 300,000 rpm (e.g., corresponding toabout 2,500 g to about 65,000 g).

Referring particularly to FIG. 15 , during an initial phase of spinning,the specimen 1001 descends within the equilibration solution 1020 whilethe equilibration solution 1020, containing the specimen 1001, descendsvia bulk motion through the separation fluid 1024 (e.g., therebydisplacing the separation fluid 1024) toward the vitrification solution1022. Referring particularly to FIG. 16 , during a subsequent phase ofspinning, the equilibration solution 1020 reaches the vitrificationsolution 1022, and the specimen 1001 passes from the equilibrationsolution 1020 into the vitrification solution 1022. Referringparticularly to FIG. 17 , during a next phase of spinning, theequilibration solution 1020 merges with the vitrification solution 1022to form a combined vitrification solution 1030 (e.g., including theequilibration solution 1020, the vitrification solution 1022, and amixed solution interface layer between the equilibration solution 1020and the vitrification solution 1022), and the specimen 1001 continues todescend through the combined vitrification solution 1030.

Referring particularly to FIG. 18 , during a final phase of spinning,the specimen 1001 rests on a meniscus 1032 of the distal air pocket 1028due to surface tension and thereby avoids contact with the relativelyhard wall of the elongate tube 1002. For example, due to a balancebetween surface tension at the interface of the combined vitrificationsolution 1030 and the distal air pocket 1028, and tension betweencombined vitrification solution 1030 and an interior wall of the taperedportion 1016, the potential buoyancy force of the distal air pocket 1016is not sufficient to break through meniscus 132. Therefore, the specimen1001 cannot penetrate the meniscus 1032.

With the specimen 1001 resting on the meniscus 1032 of the distal airpocket 1028 upon completion of spinning, the timer 126 is activated, andthe specimen container 1000 is allowed to sit in place (e.g.,stationary) in the receptacle 162 for a second predetermined exposureperiod for the specimen 1001 to be exposed to the combined vitrificationsolution 1030. The second exposure period may range from about 0.5minutes to about 2 minutes, depending on various parameters of typicalART protocols. During the second exposure period, permeation ofcryoprotectants within the combined vitrification solution 1030 into thespecimen 1001 replaces water within the specimen 1001, therebydehydrating the specimen and further infusing the specimen 1001 withcryoprotectants. Such a stage-like progression of media concentrationsavoids an excessively high initial osmotic differential that couldotherwise cause cells of the specimen 1001 to shrink too much and toorapidly as the water leaves the cells at a rate faster than thecryoprotectants can enter the cells.

Owing to a preloaded state of the equilibration solution 1020 and thevitrification solution 1022 within the specimen container 1000, aspecimen 1001 can be prepared for vitrification within a single,isolated environment (e.g., the lumen of the specimen container 1000)without being exposed to contamination, mechanical damage (e.g., from amicropipette or other specimen holding or fluid delivery device), orother accidental mishandling that may otherwise occur when a containerthat houses a specimen is accessed multiple times to deliver and removevarious processing mediums or when a specimen is moved to variouscontainers (e.g., petri dishes, test tubes, or flask) during an ARTprocess.

In some implementations, once the second exposure period has ended, thespecimen container 1000, containing the specimen 1001, is then manuallytransferred from the receptacle 162 to a long-term low temperaturestorage structure, where the specimen 1001 can be maintained in acryogenic state for a period of up to about 20 years. In some instances,the specimen container 1000 may be stored in the long-term lowtemperature storage structure for a much shorter period (e.g., as shortas few hours).

The software algorithm 300 used to track the position of the specimen1001 may be executed on the microcontroller 130 or on a separate,external computing device (e.g., a desktop computer, a laptop, a tablet,or a single board computer) running an operating system that iselectrically coupled to the specimen processing system 100 via a dataconnection (e.g., a USB connection, an RS232 connection, or a wirelessdata connection). Referring to FIG. 19 , the software algorithm 300enters a process flow loop in which the software acquires a single colorimage from the camera feed (302), converts the image to greyscale, andstores the greyscale image in an array that holds a grey value for eachpixel of the greyscale image (304). The algorithm 300 then performs anedge detection routine on the greyscale image to detect edges (e.g., anoutline) of the specimen container 1000 and therefore define a size anda position of an area of interest with respect to a field of view of thecamera 180 in which the position of the specimen 1001 will be tracked(306).

The algorithm 300 then captures a first subsequent color image from thecamera feed (308), waits for a period of time (310), and then captures asecond subsequent color image from the camera feed (312). As the firstand second subsequent color images are captured, the images are croppedto the area of interest. The algorithm 300 also converts the first andsecond subsequent color images to greyscale, and stores the first andsecond greyscale images in an array that holds a grey value for eachpixel of the greyscale images (314). The algorithm 300 then compares thefirst and second greyscale images to each other and generates anadditional array that stores the pixilation differences between thefirst and second grayscale images as a difference image (316). Thealgorithm 300 converts luminosity data from the difference image to abinary value based on an upper constraint and a lower constraint togenerate a first binary threshold difference image (318). For example,all image data that falls between the upper and lower constraints ismaintained in the first binary threshold difference image, whereas allimage data that falls outside of the range defined between the upper andlower constraints is discarded.

The algorithm 300 then blurs the first binary threshold difference imageto remove noise and thereby generates a first blur difference image(320). The algorithm 300 again converts luminosity data from the firstblur difference image to a binary value based on an upper constraint anda lower constraint to generate a second binary threshold differenceimage with even less noise as compared to the first binary thresholddifference image (322). In this case, the binary value, upperconstraint, and lower constraint are independent of those used togenerate the first binary threshold difference image. The algorithm 300also blurs the second binary threshold difference image to furtherremove noise and thereby generate a second blur difference image (324).

The algorithm then passes the second binary threshold difference imageto an object detection routine (326) in which a specimen 1001 may beidentified in the image. If a specimen 1001 is not identified in theimage (328), then the algorithm 300 returns to the step of capturing afirst subsequent color image from the camera feed (308). If a specimen1001 is identified in the image (328), then the algorithm 300categorizes (e.g., determines) a location of the specimen 1001 andstores the location in an array of object positions (330). Using apredetermined maximum and minimum threshold, the algorithm 300identifies the specimen 1001 based on the number of pixels (e.g., for aknown camera resolution), which represents a generally circular area ofthe specimen 1001 under a known magnification within the array (332).The algorithm 300 stores a center position, a speed (based on a timeelapsed between the previously processed image and positions of thespecimen 1001 in the current and previously processed image), and adirection of the specimen 1001 in another array (334). For example, themaximum and minimum thresholds provide maximum and minimum limits countof at least partially contiguous pixels forming a generally circulararea that represents approximate geometry limits of the specimen 1001.Records of speed and position of the center position are tracked toverify the motion of at least one specimen 1001.

The algorithm 300 outputs the center position, speed, and direction dataof the specimen 1001 for further processing (336). For example, in someembodiments, the algorithm 300 outputs the data to the display screen110 for viewing by an operator (338) and to a component of themicrocontroller 130. In some embodiments, the algorithm 300 additionallyoutputs the data to a component of the external computing device via thedata connection. If the algorithm 300 has finished tracking the specimen1001 (340), then the algorithm 300 exits the process flow loop. If thealgorithm 300 has yet to finish tracking the specimen 1001 (340), thenthe algorithm 300 returns to capture a first subsequent color image fromthe camera feed (308).

Using the information from the algorithm 300, the microcontroller 130can control the rotational speed, spin direction, and acceleration ofthe platform 104 via communication with the motor assembly 140 to ensurethat the specimen 1001 is exposed to a substantially constantcentripetal force as programmed by the user, irrespective of an axialposition of the specimen 1001 within the specimen container 1000 (e.g.,a radial position of the specimen 1001 along the platform 104). Forexample, according to one or more signals transmitted by themicrocontroller 130, the platform 104 can spin about the central axis152 to exert enough centripetal force on the specimen 1001 to cause thespecimen 1001 to move along the central axis 1028 of the specimencontainer 1000 toward the distal closure 1006 according to a specifiedprotocol. The one or more signals can be used to adjust an angular speedof the platform 104 and/or a duration of one or more phases of theprotocol. Such protocol adjustments can optimize time periods ofspecimen exposure to the processing media within the specimen container1000.

In some embodiments, a specimen container that is otherwise similar tothe specimen container 1000 may itself include one or more embeddedoptical elements (e.g., one or more lenses) that enable a specimen 1001to be more clearly seen by the naked eye or visualized by the camera 180of the vision system 200 during or separate from an automated specimentracking routine carried out at the specimen processing system 100.

In some embodiments, the specimen processing system 100 is furtherequipped with one or more vibration assemblies designed to excitemovement of either or both of a specimen 1001 or fluids within aspecimen container 1000 while the specimen container 1000 is processedat the specimen processing system 100. For example, FIG. 20 illustratessuch a vibration assembly 400 that is designed to securely support aspecimen container 1000. One or more vibration assemblies 400 can berespectively installed to one or more of the processing stations 102 ofthe specimen processing system 100 in place of a respective upperbracket 160 of a processing station 102.

The vibration assembly 400 includes a base 402 that can be secured tothe platform 104 at a processing station 102 and to which the othercomponents of the vibration assembly 400 are mounted. The vibrationassembly 400 further includes a mounting platform 404 that is formed tosupport a specimen container 1000 and that is movable (e.g., suspendedin free space) with respect to the base 402. For example, the vibrationassembly 400 further includes two frames 406 along which the mountingplatform 404 can move laterally and longitudinally, two dynamic spacers408 (e.g., springs or other members made of compliant materials) thatlimit excessive outward movement due to centripetal force duringspinning, and an adjustable stop 410 that permits some free movementagainst the dynamic spacers 408 without the need to rigidly attach themounting platform 404 to the base 402. At least a central portion of themounting platform 404 is made of an optically transparent material toallow focused light from the vision system 200 to pass through andilluminate the specimen 1001 within the specimen container 1000. Thevibration assembly 400 also includes opposed restraining clamps 412 thatcan clamp the specimen container 1000 to the mounting platform 404.

The vibration assembly 400 further includes a motor 414 that vibratesthe mounting platform 404 along an x axis and a motor 416 that vibratesthe mounting platform 404 along ay axis. The motors 414, 416 may beactivated via electrical signals received from the electrical contacts156 within the housing 106. The motors 414, 416 may be activatedsimultaneously or at different times to achieve a desired movementdirection. A drive voltage of the motors 414, 416 may also be adjustedto change vibration frequencies of the motors 414, 416.

In some embodiments, a specimen processing system that is similar inconstruction and function to the specimen processing system 100 may befurther equipped with features for cutting and subsequently sealing aspecimen container 1000. For example, in some examples, there may be aneed to cut excess length from the specimen container 1000 after thespecimen 1001 has been processed and is disposed at a distal end of thespecimen container 1000. FIG. 21 illustrates a cut-and-seal station 502of a specimen processing system 500 at which a specimen container 1000can be simultaneously cut and sealed (e.g., via a heat seal, anultrasonic seal, or a crimp) prior to a distal storage portion 503 ofthe specimen container 1000 being placed in a low temperature substance501. In some embodiments, a specimen container 1000 may be cut andsealed in two separate operations. For example, FIG. 22 illustrates acutting station 602 of a specimen processing system 600 at which aspecimen container 1000 can first be cut and then be automaticallysealed, capped, or plugged using dedicated equipment that is part of thespecimen processing system 600 prior to a distal storage portion 603 ofthe specimen container 1000 being placed in a low temperature substance601.

While the above-discussed specimen processing system 100, specimenprocessing system 500, specimen processing system 600, specimencontainer 1000, vision system 200, and vibration assembly 400 have beendescribed and illustrated as including components with certaindimensions, sizes, shapes, materials, and configurations, and withrespect to the software algorithm 300, in some embodiments, specimenprocessing systems, specimen containers, vision systems, vibrationassemblies, and software algorithms that are otherwise substantiallysimilar in structure and function to the above-discussed embodiments mayinclude one or more components with different dimensions, sizes, shapes,materials, and configurations or one or more different process flowsteps.

For example, while the specimen processing system 100, the vision system200, and the algorithm 300 have been described and illustrated withrespect to tracking one specimen 1001 within a specimen container 1000,in some embodiments, a specimen processing system that is substantiallysimilar in construction and function to the specimen processing system100 may be operated with an algorithm that is designed to track morethan one specimen 1001 within the same specimen container 1000 during aspecimen processing protocol.

Accordingly, other embodiments are within the scope of the followingclaims.

What is claimed is:
 1. A specimen processing system, comprising: a platefor supporting a specimen system, the specimen system comprising aspecimen container and a specimen contained therein; a camera disposedabove the plate and configured to generate images of the specimensystem; a light source disposed beneath the plate for radiating lighttowards the plate; a light stop for blocking a portion of the light fromreaching the specimen system to produce darkfield illumination of thespecimen at the camera; a rotatable platform to which a processingstation is secured for applying a centripetal force to the specimen tocause the specimen to move within the specimen container; and one ormore processors electronically coupled to the camera, the one or moreprocessors configured to convert the images from color to greyscale andto track a position of the specimen within the specimen container duringa vitrification protocol based on the images.
 2. The specimen processingsystem of claim 1, further comprising an adjustable lens for focusingthe light onto the specimen system.
 3. The specimen processing system ofclaim 1, further comprising the processing station, wherein theprocessing station locates the camera.
 4. The specimen processing systemof claim 3, wherein the processing station defines a receptacle adjacentthe plate for positioning the specimen container.
 5. The specimenprocessing system of claim 3, wherein the processing station comprises amount for selectively positioning the camera at the processing station.6. The specimen processing system of claim 1, wherein the one or moreprocessors are further configured to remove noise from the images. 7.The specimen processing system of claim 1, wherein the one or moreprocessors are further configured to detect an object corresponding tothe specimen in the images.
 8. The specimen processing system of claim1, wherein the one or more processors are further configured todetermine parameters including one or more of a position, a speed, and adirection of the specimen as the specimen moves within the specimencontainer.
 9. The specimen processing system of claim 8, wherein the oneor more processors are configured to output one or more of theparameters.
 10. The specimen processing system of claim 9, wherein thespecimen processing system comprises a motor that can adjust movement ofthe rotatable platform based on one or more of the parameters.
 11. Thespecimen processing system of claim 1, wherein the light stop isarranged to block the portion of the light from reaching a central axisof the specimen container such that edges of the specimen remain visibleto produce darkfield illumination at the camera.
 12. The specimenprocessing system of claim 1, wherein the light source comprises aplurality of light-emitting diodes.
 13. The specimen processing systemof claim 1, wherein the camera is configured to scan an identificationlabel of the specimen container.
 14. The specimen processing system ofclaim 1, wherein the one or more processors are configured to trackrespective positions of a plurality of specimens within the specimencontainer based on the images during the vitrification protocol.
 15. Thespecimen processing system of claim 1, further comprising a vibrationassembly configured to direct movement of the specimen within thespecimen container during the vitrification protocol.
 16. The specimenprocessing system of claim 1, further comprising a cutting stationconfigured to cut and release a distal portion of the specimen containerwith the specimen contained therein following completion of thevitrification protocol.
 17. The specimen processing system of claim 1,wherein the specimen comprises a reproductive specimen.
 18. A method ofprocessing a specimen within a specimen container, the methodcomprising: generating images of the specimen within the specimencontainer at a camera disposed above a plate supporting the specimencontainer; directing light towards the plate from a light sourcedisposed beneath the plate; blocking a portion of the light fromreaching the specimen with a light stop to produce darkfieldillumination of the specimen at the camera; applying a centripetal forceto the specimen to cause the specimen to move within the specimencontainer by rotating a platform to which a processing station issecured; converting the images from color to greyscale at one or moreprocessors in electronic communication with the camera during avitrification protocol; and tracking a position of the specimen withinthe specimen container based on the images at the one or moreprocessors.
 19. The method of claim 18, further comprising focusing thelight onto the specimen at an adjustable lens.
 20. The method of claim18, further comprising locating the camera at the processing station.21. The method of claim 20, further comprising positioning the specimencontainer within a receptacle of the processing station that is adjacentthe plate.
 22. The method of claim 20, further comprising selectivelypositioning a mount supporting the camera at the processing station. 23.The method of claim 18, further comprising removing noise from theimages at the one or more processors.
 24. The method of claim 18,further comprising detecting an object corresponding to the specimen inthe images at the one or more processors.
 25. The method of claim 18,further comprising determining, at the one or more processors,parameters including one or more of a position, a speed, and a directionof the specimen as the specimen moves within the specimen container. 26.The method of claim 25, further comprising outputting one or more of theparameters from the one or more processors.
 27. The method of claim 26,further comprising adjusting movement of the platform based on one ormore of the parameters via a motor.
 28. The method of claim 18, furthercomprising blocking the portion of the light from reaching a centralaxis of the specimen container such that edges of the specimen remainvisible to produce darkfield illumination at the camera.
 29. The methodof claim 18, wherein the light source comprises a plurality oflight-emitting diodes.
 30. The method claim 18, further comprisingscanning an identification label of the specimen container at thecamera.
 31. The method of claim 18, further comprising trackingrespective positions of a plurality of specimens within the specimencontainer based on the images at the one or more processors during thevitrification protocol.
 32. The method of claim 18, further comprisingdirecting movement of the specimen within the specimen container at avibration assembly during the vitrification protocol.
 33. The method ofclaim 18, further comprising cutting and releasing a distal portion ofthe specimen container, with the specimen contained therein, followingcompletion of the vitrification protocol at a cutting station.
 34. Themethod of claim 18, wherein the specimen comprises a reproductivespecimen.